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The blastema and epimorphic regeneration in mammals

Article  in  Developmental Biology · December 2017

DOI: 10.1016/j.ydbio.2017.08.007

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 Developmental Biology 433 (2018) 190–199

Contents lists available at ScienceDirect

Developmental Biology
journal homepage: www.elsevier.com/locate/developmentalbiology

Review article

The blastema and epimorphic regeneration in mammals MARK

a,⁎ b,⁎
Ashley W. Seifert , Ken Muneoka
a
Department of Biology, University of Kentucky, Lexington, KY 40506, United States
b
Department of Veterinary Physiology and Pharmacology, Texas A & M University, College Station, TX 77843, United States

A BS T RAC T

Studying regeneration in animals where and when it occurs is inherently interesting and a challenging research
topic within developmental biology. Historically, vertebrate regeneration has been investigated in animals that
display enhanced regenerative abilities and we have learned much from studying organ regeneration in
amphibians and fish. From an applied perspective, while regeneration biologists will undoubtedly continue to
study poikilothermic animals (i.e., amphibians and fish), studies focused on homeotherms (i.e., mammals and
birds) are also necessary to advance regeneration biology. Emerging mammalian models of epimorphic
regeneration are poised to help link regenerative biology and regenerative medicine. The regenerating rodent
digit tip, which parallels human fingertip regeneration, and the regeneration of large circular defects through
the ear pinna in spiny mice and rabbits, provide tractable, experimental systems where complex tissue
structures are regrown through blastema formation and morphogenesis. Using these models as examples, we
detail similarities and differences between the mammalian blastema and its classical counterpart to arrive at a
broad working definition of a vertebrate regeneration blastema. This comparison leads us to conclude that
regenerative failure is not related to the availability of regeneration-competent progenitor cells, but is most
likely a function of the cellular response to the microenvironment that forms following traumatic injury. Recent
studies demonstrating that targeted modification of this microenvironment can restrict or enhance regenerative
capabilities in mammals helps provide a roadmap for eventually pushing the limits of human regeneration.

1. Introduction
While regeneration refers to the replacement of a lost body part, the
term also describes the repeated replacement of skin cells or shark's
teeth, the generation of a whole organism from part of an embryo, or in
the broadest sense, the process of embryogenesis from a fertilized egg
(the regeneration of an organism from a genome). These and other
examples are classically typed as either physiological (homeostatic) or
reparative regeneration, where physiological regeneration describes the
regular replacement of cells and tissues during homeostasis and aging
(e.g., epidermis, blood, shark's teeth, etc.), and reparative regeneration
occurs in response to injury (Morgan, 1901). Homeostatic regeneration
is a ubiquitous property of vertebrates until that time when cells can no
longer replace themselves and tissues and organs begin to fail. When
considered in this context, all vertebrates exhibit a capacity for tissue
regeneration. The enigma for regeneration biologists is that vertebrate
species appear to be distributed along a continuum of reparative
regenerative ability. As a biological problem, we still do not understand
why some adult vertebrates can regenerate organs in response to
damage while others heal similar injuries with scar tissue. Viewed as a
clinical problem, although all tissues of the human body display some
level of homeostatic regenerative ability, complex tissues and organs do
not generally regenerate in response to injury. Can complex tissue
regeneration be stimulated in non-regenerating species? These ques-
tions are the nexus of regenerative biology and medicine.
The cell is the basic unit of tissue regeneration. Accordingly,
regeneration can occur at multiple levels of biological organization.
In one dimension the smallest injury requires repair of a single cell
(e.g., a severed axon). In another, severe trauma requires replacement
of an entire organ de novo through the coordinated morphogenesis of
millions of cells into distinct tissue types (e.g., a limb following
amputation). Somewhere in between is the regeneration of variably
complex structures with multi-cellular architecture and multiple func-
tional units (e.g., ear punch, spinal cord resection, skeletal fracture,
etc.). Regeneration in all of these examples is intimately tied to the
regulated activation, coordinated growth, and differentiation of local
cells, be they adult stem cells, or differentiated cells that undergo de-
differentiation or reprogramming. How are resident cells activated and
coordinated to regenerate organs?
Generally speaking, after blood loss is stemmed and an organism

⁎
Corresponding authors.
E-mail addresses: awseifert@uky.edu (A.W. Seifert), KMuneoka@cvm.tamu.edu (K. Muneoka).

http://dx.doi.org/10.1016/j.ydbio.2017.08.007
Received 2 June 2017; Received in revised form 28 July 2017; Accepted 4 August 2017
Available online 25 December 2017
0012-1606/ © 2017 Elsevier Inc. All rights reserved.
A.W. Seifert, K. Muneoka
mounts defensive action from infection, local cells accumulate and
produce new tissue. Among vertebrates, the process of regenerating a
replacement organ in situ requires cell proliferation and is referred to
as epimorphic regeneration (Morgan, 1901). This distinguishes it from
regeneration that involves the reorganization of existing cells at the
wound site to restore normal patterning prior to growth (i.e., mor-
phallaxis). Appendage regeneration in salamanders and newts is the
classic example of a complete and complex epimorphic response (Goss,
1969; Wallace, 1981). While amputating one lobe of the liver will also
stimulate cell (hepatocyte) proliferation to restore organ function, the
lobe itself does not regenerate. What distinguishes epimorphic regen-
eration from disorganized tissue regeneration is that a regenerating
limb or tail forms from a transient proliferative mass called a blastema.
While a similar response is not usually observed following appendage
amputation in mammals, there do exist a few mammalian examples of
epimorphic regeneration in which a blastema forms, and these can
instruct the likelihood of enhancing regenerative capabilities in hu-
mans.
In this review we focus our attention on digit tip and ear hole
regeneration as two examples of blastema-based epimorphic regenera-
tion in mammals (Gawriluk et al., 2016; Joseph and Dyson, 1966;
Muneoka et al., 2008). As described below, these two mammalian
models present unique opportunities to discover the mechanisms that
stimulate a regenerative response to injury in lieu of fibrotic healing. In
focusing on the digit and ear pinna we do not discuss the broader
regenerative abilities of the spiny mice primarily because full-thickness
skin regeneration is not mediated by a blastema (Seifert et al., 2012;
Seifert and Maden, 2014). Similarly, we have chosen to exclude the
annual shedding and regrowth of deer antlers which represents an
extreme example of physiological regeneration (Kierdorf et al., 2009).
While some authors have suggested deer antler regeneration is
blastema-mediated (Li, 2012) studies have shown that antler regen-
eration is nerve-independent and the antler bud displays character-
istics that are distinct from an epimorphic blastema (Kierdorf et al.,
2007, 2009). Thus, while fascinating, in light of these differences and
the fact that antler regeneration does not occur in response to injury, it
is difficult to find parallels with other blastema-mediated regenerative
responses in mammals. Ultimately, studying different types of regen-
eration reveals the diversity of regenerative processes. In our view,
however, understanding the mechanisms that stimulate and regulate
blastema formation is the key to moving from a deconstructive to
constructive study of regeneration.
2. The vertebrate regeneration blastema
Several factors have emerged from the vertebrate regeneration
literature as key components in all known examples of blastema-based
epimorphic regeneration. These include: (1) formation of a specialized
wound epidermis that functions to attract blastemal cells and maintain
cell proliferation (Globus et al., 1980b; Thornton, 1957a; Thornton and
Steen, 1962; Thornton and Thornton 1965), (2) dependence on
innervation and exposure to nerve or Schwann cell secreted factors
(Farkas et al., 2016; Kumar et al., 2007; Mescher et al., 1997; Mullen
et al., 1996; Singer, 1952), (3) formation of a pro-regenerative
extracellular matrix (Calve et al., 2010; Gawriluk et al., 2016;
Mailman and Dresden, 1979; Marrero et al., 2017; Onda et al., 1991;
Satoh et al., 2012; Seifert et al., 2012; Tassava et al., 1996; Vinarsky
et al., 2005), (4) deployment of major developmental signaling path-
ways (rev. in Stoick-Cooper et al., 2007), (5) physical interaction of
cells from antonymic positions in three-dimensional space (Carlson,
1974; Cook and Seifert, 2016; Lheureux, 1975a, 1975b), (6) recogni-
tion of uninjured versus new tissue and thus level-specific replacement
of appropriate tissue to generate a complete organ and (7) a depen-
dence on macrophages to initiate regeneration (Godwin et al., 2013;
Petrie et al., 2014; Simkin et al., 2017). Together, these features
contribute to, and support, blastema formation, without which regen-
191
Developmental Biology 433 (2018) 190–199
eration will not occur. Against the backdrop of these features
epimorphic regeneration involves two major transformations in re-
sponse to injury: 1) mature tissue into a blastema and 2) a blastema
into a regenerated organ. Viewed in this light, the blastema is the link
between healing and morphogenesis.
2.1. How to broadly define a blastema?
Blastema: From Greek blastein – to sprout; ma – result of action
A blastema is a heterogeneous cell mass that through migration and
proliferation transiently forms at the injury site and undergoes
morphogenesis to form the missing organ. We use morphogenesis as
it refers to organogenesis (i.e., organ development during embryonic
development), but occurring in the adult form. The heterogeneous
mass of cells is necessarily covered by epidermis (termed the wound
epidermis) and thus the definition of a blastema includes this
ectoderm-derived component. Historically, a vertebrate blastema was
often defined as a mass of pluripotent cells where blastemal cells would
contribute to all the of the regenerated structures. In reality, classic and
modern lineage tracing techniques have shown that blastemal cells
tend to respect developmental lineages during normal regeneration
(rev. in Monaghan and Maden, 2013), although some evidence
suggests that connective tissue fibroblasts over-contribute progeny to
the blastema (Dunis and Namenwirth, 1977; Kragl et al., 2009;
Muneoka et al., 1986). The blastema has also been classically defined
by its developmental potential, i.e., what it transforms into distin-
guishes the blastema from any other mass of undifferentiated cells
(e.g., tumor, granulation tissue, etc.). While this may be appropriate
during development (nephrogenic blastema) or during salamander
limb regeneration (limb blastema) where regeneration routinely oc-
curs, this definition is problematic in approaching translational issues
associated with regenerative failure. In this regard, it is important to
modify this classic definition and try to define a blastema by functional
attributes that are essential for establishing its’ developmental poten-
tial, rather than applying a descriptor of its ultimate developmental
fate. A broad definition of this type allows us to identify key properties
of a blastema that promote the transition from healing to morphogen-
esis and thus provides a basis for comparison across species or across
distinct injury responses within a species. This conceptual framework is
particularly useful when examining epimorphic regeneration in mam-
mals where variation in regenerative ability exists between closely
related species (ear pinna) and within an individual organ (digit). In
these instances, the presence or absence of a blastema can help provide
an explanation for regenerative success or failure.
In animals that possess regenerative capabilities, local amputation
initiates the first transformation from mature tissue into a transient
undifferentiated proliferative phase (blastema) that is followed by the
second transformation where morphogenesis and re-differentiation
replace the missing structures. The first transformation is a specialized
wound healing response that ends with the formation of the blastema
(Fig. 1). This can be distinguished from a non-regenerative repair
response where re-epithelialization is followed by reconstitution of a
mature basement membrane, wound contraction, and the deposition of
a densely layered fibrous scar tissue that defines regeneration-incom-
petence. During a regenerative response where the first transformation
ends with the formation of a small mass of undifferentiated proliferat-
ing cells, the second transformation involves proliferative expansion of
this cell population, patterning, and ultimately the orderly differentia-
tion into the multitude of cell types that make up the tissues of the
replacement structure. In the context of regenerative failure, these two
transforming events are generally viewed as distinct and independent
biological problems. In other words, the study of mammalian regen-
eration involves two distinct and historically separate fields of inquiry:
wound healing, which is largely focused on tissue healing that is non-
regenerative and resolves in the formation of scar tissue, and tissue
development, which is largely studied during embryogenesis. In
A.W. Seifert, K. Muneoka Developmental Biology 433 (2018) 190–199

Fig. 1. Schematic depicting formation of the regenerating mouse digit tip blastema. (A) Cartoon of the mouse digit tip. The P3 bone (blue) is shown as triangular with a proximal base
that contains a bone marrow region (white) and tapers to the distal tip. The bone marrow is highly vascularized (not shown) and hypocellular (red). Epidermis (black) and connective
tissue cells (green) surround the P3 bone. Amputation of the digit tip does not damage the bone marrow. (B) Following amputation of the distal bone the epidermis heals onto the
periosteum of the stump. The distal bone stump contains dead bone (red stars). (C) Histolysis of the stump is mediated by multinucleated osteoclasts (yellow) that erode the bone and
creates a secondary amputation plane. This phase is associated with increased cell numbers in the connective tissue and bone marrow. (D) Once the secondary amputation is complete,
the wound epidermis closes through the region of degraded bone and a blastema forms. Cells from the bone marrow and surrounding connective tissue participate in blastema
formation. The P3 level that regenerates is proximal to the original level of digit amputation.

regeneration competent models, the blastema sits at this interface
making the study of its formation critical for understanding how
regeneration is controlled.
Whether or not morphogenesis is an emergent property of the
blastema or something that must be stimulated is unknown. If the
former, then discovering the ability to create a regeneration blastema
may be the key to stimulating successful regeneration where it does not
naturally occur (Goss, 1980). Focusing on the first transformation, it
remains unresolved if pro-regenerative signals distinct from general-
ized injury signals exist to stimulate blastema formation (Sugiura et al.,
2016; Tassava and Mescher, 1975). One way or another, the early
response to injury during epimorphic regeneration involves acute
inflammation that is concurrent with re-epithelialization and formation
of the wound epidermis (Gauron et al., 2013; Godwin et al., 2013; Love
et al., 2013; Mescher and Neff, 2005; Simkin et al., 2017). As the
inflammatory response resolves, local histolytic activity, re-innerva-
tion, and extracellular matrix production facilitates the migration and
accumulation of cells beneath the wound epidermis to form the nascent
blastema (Calve et al., 2010; Singer, 1952; Vinarsky et al., 2005). In
amphibians and fish, cells of the regenerating limb/fin are derived from
multiple tissue types and lineage restricted progenitor cells have been
clearly identified (Knopf et al., 2011; Kragl et al., 2009; Singh et al.,
2012; Tu and Johnson, 2011). In addition to the variety of lineage
restricted progenitor cells that make up the blastema, there is evidence
that fibroblastic cells of the interstitial connective tissue are an
important cell source for the blastema (Muneoka et al., 1986; Tank
and Holder, 1979). The connective tissue of the dermis has been
studied most extensively using cell markers coupled with skin trans-
plantation. Such tissue grafting studies show that cells of the dermis
over-contribute to the blastema, and that these cells participate in
regenerating skeletal limb tissues (e.g., bone, cartilage, tendons) as well
as re-forming the dermis (Dunis and Namenwirth, 1977; Kragl et al.,
2009; Muneoka et al., 1986). Since there are a number of distinct cell
types in dermal connective tissue, e.g., vascular, and perivascular cells,
and heterogeneity among fibroblasts (Driskell et al., 2013; Rinkevich
et al., 2015) it remains to be demonstrated whether one or all of these
cell types are multipotent. Nevertheless, since there are no skeletal
structures in the dermis and yet cells from the dermis can contribute to
regenerated bone and cartilage, the evidence clearly indicates the
existence of a multipotent cell type within the dermis. Unfortunately,
the heterogeneity and plasticity of blastemal cells from tissue-specific
lineages does not aid in the task of defining the blastema generally,
rather, these cellular properties are a function of the specific tissue
injured. Instead, a useful definition relies on the fact that some fraction
of blastemal cells is stimulated to re-enter the cell cycle and undergo
cell cycle progression and division (Globus et al., 1980a; Hay and
Fischman, 1961; Tomlinson and Barger, 1987). While the persistence
of active cycling cells can be used to identify the blastema, this alone
hardly differentiates a blastema from a tumor. Therefore, the presence
of proliferating cells must be used in combination with other factors.
Given that uncontrolled growth is a characteristic of tumor cells and
controlled growth is a characteristic of blastemal cells, comparative
profiling of cycling tumor cells and blastemal cells may identify a panel
of markers specific to cycling blastemal cells. Until these markers are
found, however, one must look to other factors to uniquely define a
blastema.
It has long been recognized that a wound epidermis forms atop the
blastema after re-epithelialization and is required to direct growth of
the blastema, maintain cell proliferation, and prevent premature
differentiation (Globus et al., 1987, 1980b; Thornton, 1960). Studies
that denude the blastema of the wound epidermis delay regeneration
until re-epithelialization occurs again because the wound epidermis
continually reforms after injury (Thornton, 1957b). In this regard, the
wound epidermis is an integral part of the blastema and essential for
successful regeneration. Once formed, the wound epidermis is further
modified by the action of re-growing axons (Endo et al., 2004; Satoh
et al., 2008). In salamanders and newts, innervation helps transition
the new epidermis into a signaling center called the apical epithelial
cap (AEC). This interaction with re-growing nerves is required for
blastema formation and distinguishes the AEC from the neoepidermis
that forms atop non-blastema-based regenerating skin wounds (Endo
et al., 2004; Satoh et al., 2008). Several molecular markers have been
identified that demarcate this specialized epidermis from epidermis
located proximal to the injury or from neoepidermis covering tissue
that does not form a blastema (Gawriluk et al., 2016; Han et al., 2001;
Mullen et al., 1996; Satoh et al., 2008). In addition to molecular
markers, the wound epidermis is morphologically distinct from the
normally stratified epithelium typical of adult skin. As regeneration
progresses reformation of the mature basement membrane beneath the
wound epidermis is delayed and its absence is a useful indicator of the
wound epidermis (Gawriluk et al., 2016; Neufeld and Day, 1996;
Neufeld et al., 1996; Seifert et al., 2012). Although further molecular
and functional characterization of the wound epidermis is necessary,
current markers and morphological features can identify a wound
epidermis as a fundamental component of the blastema.
Lastly, there is increasing evidence that the blastemal extracellular
matrix is a key component of regeneration, although its role as a
facilitator, regulator or passive support structure remains unclear
(Calve et al., 2010; Gawriluk et al., 2016; Mailman and Dresden,
1979; Marrero et al., 2017; Onda et al., 1991; Satoh et al., 2012;
Seifert et al., 2012; Tassava et al., 1996; Vinarsky et al., 2005). Early
studies of salamander and newt blastemas detected increased levels of
matrix proteins associated with cell migration and proliferation (e.g.,
fibronectin and tenascin-C) (Onda et al., 1991; Tassava et al., 1996). In
vitro work examining newt myotubes on fibronectin, tenascin-c and
hyaluronic acid showed these proteins could directly regulate cell
behavior (Calve et al., 2010; Calve and Simon, 2012). More recent work
in mammals (see below) suggests that the extracellular composition of
the blastema can itself help define this transient structure when
compared to non-regenerating tissues where a blastema does not form
(Gawriluk et al., 2016; Marrero et al., 2017; Seifert et al., 2012). All told,
while we have an incomplete definition of a blastema, the indicators
outlined above do provide a conceptual foundation to identify pro-
regenerative factors from cross-species and inter-injury comparisons.

192
A.W. Seifert, K. Muneoka
3. Two mammalian blastemas – digits and ears
As we continue to discover fundamental mechanisms underlying a
regenerative response to injury, the importance of studying natural
examples of reparative regeneration in mammals is undeniable. Adult
regeneration models in mammals, while not as spectacular as limb
regeneration in urodeles, do afford researchers a comparative approach
to understand basic principles of blastema formation and morphogen-
esis. To this end, comparing regenerating and non-regenerating
injuries should help expand our ability to define the regeneration
blastema. Moreover, putting phylogenetic considerations aside, impor-
tant physiological differences between mammals and traditional re-
generation models like salamanders, newts and zebrafish (e.g., home-
othermy versus poikilothermy, high versus low metabolic rates, etc.)
make it vital to understand how tissues can regenerate against the
backdrop of mammalian physiology. As another example, although an
innate immune response to injury is an ancient weapon against
infection, it seems unlikely that this response is exactly the same in
newts as it is in a mouse or human. Below we offer two models for
epimorphic regeneration in mammals where steady progress is con-
tributing to our understanding of blastema formation, its definition,
and more broadly, to the differences between a regenerative or fibrotic
response to injury.
3.1. Digit tip regeneration
The regeneration of human fingertips is well documented in the
clinical literature (Illingworth, 1974) and parallels between digit tip
regeneration in mice and fingertip regeneration in humans have peaked
interest in the feasibility of human regeneration (Muneoka et al.,
2008). The regenerating mouse digit has become an important experi-
mental model to explore fundamental mechanisms of mammalian
regeneration and to test strategies aimed at enhancing regenerative
capabilities (Dawson et al., 2016). The mouse digit tip regenerates at all
stages of development, including adults, and the process involves the
formation of a transient blastema. The regeneration competent region
consists of the terminal or third phalangeal element (P3), a structurally
unique bone that has the shape of a flattened cone with a wide basal
region that contains a bone marrow cavity and a tapered distal tip
(Fig. 2A-B). The second phalangeal element (P2) articulates with the
base of P3 forming the P2/P3 joint (Fig. 2B). The P3 element is encased
by the nail organ which is required for the regenerative response
(Takeo et al., 2013), and a thin layer of loose connective tissue
consisting of fibroblasts, vasculature and nerves separate the P3 bone
from the nail epidermis. Amputation through the distal half of P3
initiates the regenerative response, while amputation at the base of P3
undergoes a healing response with no evidence of regeneration
(Neufeld and Zhao, 1995). After amputation, the continuously growing
nail encases the regenerate, initially appearing as a truncated out-
growth before eventually conforming to the contour of the blastema
(Fig. 2C-E). Internal to the nail the regeneration response involves a
series of interconnected and interdependent processes that transforms
the differentiated stump tissues into a blastema that undergoes
morphogenesis to form replacement tissues.
Unlike some other regeneration models, the digit amputation
wound does not undergo a rapid re-epithelialization response, instead
the epidermis initially heals onto the lateral regions of the amputated
stump bone and initiates a histolytic phase (Fig. 2F) that is dominated
by the recruitment of osteoclasts and the progressive degradation of the
stump bone (Fernando et al., 2011; Simkin et al., 2015b). This
histolytic phase correlates with the acute inflammatory response, and
the recruitment of the monocyte/macrophage lineage cells that fuse to
form large multinucleated osteoclasts (Lampiasi et al., 2016) that
degrade the stump bone (Fig. 2G). Once the stump bone is degraded,
the epidermis migrates through the region of degraded bone that
defines a new amputation level, and the blastema forms distal to the
193
Developmental Biology 433 (2018) 190–199
stump (Fig. 2H). Wound closure over the stump tissue can be induced
by use of a cyanoacrylic wound dressing, and in this case the bone
degradation phase is inhibited and a small blastema forms distal to the
original amputation (Simkin et al., 2015b). Alternatively, experimen-
tally enhancing the period of bone degradation causes greater stump
bone degradation and results in blastemas of a larger size (Sammarco
et al., 2015). Thus, there appears to be a relationship between the
extent of bone degradation and blastema size, suggestive of a relation-
ship between tissue histolysis and progenitor cell availability. Blastema
maturation is characterized by the onset of skeletal differentiation and
progresses in a proximal to distal sequence (Fig. 2H-J). The regenerat-
ing new bone forms by direct ossification with osteoblasts secreting
osteoid in a pericellular manner to rapidly form woven bone. The use of
µCT imaging has become an important tool to study the regeneration of
bone during digit tip regeneration. Bone length and volume can be
tracked and quantitated in vivo and soft tissue changes can be
quantitated using contrasting reagents (Fig. 2K-N).
The question of how the digit blastema forms is key to under-
standing both regenerative success and regenerative failure, and is an
important step for addressing the question of why most mammals
studied to date have limited regenerative capabilities. Since a blastema
can form following digit tip amputation, it is reasonable to ask why a
blastema does not form at other amputation levels. The digit blastema
can be defined based on transiently expressed characteristics asso-
ciated with its formation, and for a more stringent definition, based on
a subset of characteristics that are experimentally shown to be
functionally linked to successful regeneration. An approach of this
kind reflects the general criteria outlined above while also potentially
expanding our definition of the blastema. One example is the composi-
tion of the extracellular matrix (ECM) produced by blastema cells.
Collagen 3 is a minor component of the digit prior to amputation, but
becomes a prominent component of the blastema ECM before return-
ing to baseline levels after differentiation (Marrero et al., 2017; Simkin
et al., 2015b). The cells that produce collagen 3 have been identified as
a subset of digit fibroblasts, called fibroblast reticular cells, and they
display an enhanced proliferative response during blastema formation
(Marrero et al., 2017). Collagen 3 production by digit fibroblasts is
induced in vitro by multi-TNF receptor activation (Katakai et al., 2008;
Marrero et al., 2017) suggesting that digit fibroblasts specifically react
to inflammation by producing a provisional matrix that supports a
regenerative response. Although functional studies on the role of
collagen 3 in digit blastema formation are currently lacking, the data
are consistent with the conclusion that a collagen 3 rich matrix
supports mammalian regeneration. A more comprehensive analysis
of the extracellular environment in regenerative-competent P3 ampu-
tations compared to regeneration-incompetent P2 amputations will
help resolve how matrix proteins contribute to blastema formation.
A number of studies have identified major developmental pathways
important for digit regeneration through functional analysis. These
include multiple secreted factor signaling pathways including BMP (Yu
et al., 2010), WNT (Lehoczky and Tabin, 2015; Takeo et al., 2013,
2016), PDGF (Johnston et al., 2016), Oncostatin M (Johnston et al.,
2016), CXCL12 (Lee et al., 2013), and VEGF (Yu et al., 2014). Some of
these signaling factors are regulated in the blastema microenvironment
and control cell proliferation, cell migration or cell differentiation
(Lehoczky, 2016; Simkin et al., 2015a). A series of recent studies have
identified interactions linking the inflammatory response, angiogen-
esis, and re-epithelialization to the control of oxygen availability during
the transformation from stump to blastema. Initial studies identifying
the central region of the digit blastema as avascular echoed similar
accounts from descriptive studies of the salamander limb blastema
(Fernando et al., 2011; Mescher, 1996; Peadon and Singer, 1966; Said
et al., 2004). The avascular character of the digit blastema correlates
with reduced expression of Vegfa and up-regulated expression of the
potent anti-angiogenic factor Pedf, during early regeneration stages in
neonates (Muneoka et al., 2008; Yu et al., 2014). Experimental
A.W. Seifert, K. Muneoka Developmental Biology 433 (2018) 190–199

Fig. 2. Digit tip regeneration. (A) The distal region of the adult digit tip is comprised of the distal tip of the P3 skeletal element surrounded by a layer of connective tissue and the nail
plate (n). The plane of amputation is indicated by the solid line. (B) Sagittal section through an unamputated P3 element shows the triangle shaped bone (p3) that contains a proximal
bone marrow cavity (bm) and is enclosed by the nail (n). The P3 element articulates with the second phalangeal element (p2) at the P2/P3 joint. The plane of amputation is indicated by
the solid line. (C) External view of a regenerating digit tip at 7 days post-amputation (dpa) showing distal elongation of the nail plate (n) with no remarkable change of the stump bone.
(D) External view of a regenerating digit tip at 12 dpa showing outgrowth of a prominent digit blastema (b) surrounded by the elongating nail (n). (E) External view of a regenerating
digit tip at 17 dpa showing a regenerate that has the general shape of the terminal phalanx. Vasculature associated with ossification is apparent proximal to the distal blastema. (F)
Mallory's triple-stained section of a regenerating digit at 7 dpa showing the absence of wound closure and epidermal attachment to the lateral bone surface. Pitting of the stump bone
surface (p) is observed at this stage. (G) TRAP staining of a 7 dpa regenerate identifies multinucleated osteoclasts (*) associated with the amputated stump bone. (H) Malory's triple-
stained section of a 12 dpa digit showing the distal undifferentiated blastema (b). (I) Mallory's triple-stained regenerate at 17 dpa showing the distal stump with newly differentiated
woven bone (wb) that is capped with undifferentiated blastema cells. (J) At 28 dpa the distal P3 element is regenerated with an interlacing network of new woven bone (wb) that is
histologically distinct from the cortical bone of the stump. (K-M) µCT imaging of regenerating digits stained with a soft-tissue contrast agent (alcoholic iodine) to make the epidermis
radio-opaque and enable analyses of soft tissue versus bone. (K) At 10 dpa the distal cortical bone is eroded and the bone marrow cavity is contiguous with the distal digit blastema (b).
(L) At 17 dpa the regenerated woven bone (wb) appears as a loose network of bony tissue. (M) At 28 dpa the regenerated woven bone (wb) has a mottled appearance and is distinct from
the cortical bone of the stump. (N) Graph showing the normalized longitudinal lengths of the digit tip (solid boxes) and the P3 bone (open boxes) during the regeneration process. The
arrow highlights the transition between bone degradation, blastema formation and osteogenic differentiation of new bone. Both total digit and P3 length regenerates to normal levels
within 3–4 weeks. (O) Hypoxic character of the blastema is shown in a colorimetric overlay of hypoxyprobe staining (pink, O2 < 1.3%) and oxygen-stabilized FBXL5 immunostaining
(green, O2 > 6.0%) as compared to normoxic (purple) areas at 12 dpa. A, C-N from Fernando et al. (2011); B, from Han et al., 2008; O, from Sammarco et al. (2014).

treatment of digit amputations with VEGF induces precocious angio-
genesis and inhibits skeletal regeneration without inhibiting the
formation of a blastema structure, indicating that the avascular nature
of the blastema is a characteristic essential for regeneration (Yu et al.,
2014). The region of digit blastema avascularity displays a low oxygen
tension (< 1.3%) indicating a hypoxic microenvironment (Fig. 2O) and
suggestive of a relationship between avascularity and hypoxia
(Sammarco et al., 2014). When oxygen levels are experimentally
elevated by hyperbaric oxygen treatment, the hypoxic state of the
blastema is eliminated, and this was found to alter the regeneration
response by extending the period of osteoclast-mediated bone degra-
dation (Sammarco et al., 2015, 2014). While oxygen treatment does not
enhance osteoclastogenesis, it does inhibit the termination of the
osteoclast response, thus extending the period and extent of the bone
histolytic phase. In classic models of bone degradation, osteoclastogen-
esis is inhibited by production of osteoprotegrin, a secreted decoy
receptor that competes with and inhibits RANKL-RANK signaling that
stimulates osteoclastogenesis (Chen et al., 2017). In osteoblasts,
osteoprotegrin expression appears directly downstream of Hif2b
activity which is regulated via the oxygen sensing activity of prolyl
hydroxylase enzymes (Wu et al., 2015). These studies identify a
dynamic process that involves regulating angiogenesis to establish a
transient hypoxic blastema microenvironment that functions as a
regulatory switch to transition the histolytic phase prior to blastema
formation to the growth phase once the blastema has formed. It is
interesting that the pro-angiogenic microenvironment of granulation
tissue in full-thickness skin wounds causes excessive re-vascularization
that is linked to scar formation (DiPietro, 2016; Wietecha et al., 2015).
Thus, the evidence suggests that the regulation of this angiogenesis/
oxygen/hypoxia cascade is a potential evolutionary target responsible
for restricted regenerative capabilities in mammals, and also identifies
a target pathway to enhance regenerative ability of regeneration
incompetent wounds. The extent to which oxygen availability controls
blastema formation in other examples of epimorphic regeneration
awaits more general investigation.
One of the fundamental questions surrounding epimorphic regen-
eration responses concerns the source and potency of progenitor cells
that form the blastema and contribute to the regenerate. As indicated
above, while the identity of these cells may not help define a blastema
per se, the source and plasticity of blastemal cells can reveal funda-
mental aspects of how cellular phenotypes are modified during
regeneration. Lineage tracing studies in mice utilizing cell type specific
promoter-driven reporter expression are a powerful test for multi-
potency during regeneration. Not surprisingly, the epidermis has been
shown to be lineage restricted during digit tip regeneration as it is
during salamander limb regeneration (Kragl et al., 2009; Rinkevich
et al., 2011), and cell labeling studies show that cells derived from
hematopoietic stem cells do not contribute to the major structural
tissues of the regenerated digit (Rinkevich et al., 2011). Endothelial
cells and osteoblasts contribute to the blastema and regenerated digit,
and both are lineage-restricted with regard to their potency (Lehoczky
et al., 2011; Rinkevich et al., 2011). The use of promoter-specific Cre

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expression coupled with a constitutively active reporter gene to track
cell lineage has also been useful in determining whether specific cell
types change phenotype during regeneration. Such studies show that
Sox9-expressing skeletal cells, Scx-expressing tendon cells, and Tie2-
expressing endothelial cells do not undergo transdifferentiation during
regeneration (Rinkevich et al., 2011). While lineage studies have led to
the general conclusion that the digit blastema is composed of only
lineage restricted cells (Lehoczky et al., 2011; Rinkevich et al., 2011),
this generalized conclusion must be weighed against the limited
number of cell types that have been studied, and that the cell types
contributing to the regenerated digit (epidermis, endothelial cells,
osteoblasts) are lineage restricted during development. Indeed, with
the exception of hematopoietic stem cells that do not contribute to
regenerated structures, the contribution of more phenotypically labile
cells such as mesenchymal stem cells or tissue-specific stem cells
remain unknown. Current studies show that a panel of positive and
negative markers is required to identify certain multipotent progenitor
cells making it difficult to locate and trace these cells in vivo (Caplan
and Correa, 2011; Gökçinar-Yagci et al., 2015). Nevertheless, currently
available evidence indicates that a number of cell types participating in
digit regeneration are lineage restricted.
3.2. Ear pinna regeneration
The regeneration of holes made through the ear pinna has long
been recognized as another example of epimorphic regeneration in
mammals (Goss and Grimes, 1972; Vorontsova and Liosner, 1960).
The external ear pinna is a complex organ comprised of skin, adipose
tissue, skeletal muscle, vasculature, nerves, and elastic cartilage
(Fig. 3A-B). In some species, a complete hole punched through the
pinna removes portions of all of these structures and initiates a
regenerative response (Fig. 3C-E). The phenomenon was first reported
in 1953 with the observation that 1 cm holes made in rabbit ears
completely closed with new tissue including new hair follicles, skin and
cartilage (Markelova cited by Vorontsova and Liosner (1960)). Ensuing
investigations in the rabbit ear extended these observations and
provided evidence that the type of epidermis (i.e., abdominal vs. ear)
and presence of auricular cartilage were required for successful
regeneration (Goss and Grimes, 1972; Grimes and Goss, 1972;
Joseph and Dyson, 1966; Williams-Boyce and Daniel, 1980).
Interestingly, semicircular wounds or notches made at the edges of
the ear were reported to initiate, but not complete regeneration
(Williams-Boyce and Daniel, 1980). More recently, work with two
species of wild African spiny mouse (Acomys kempi and A. percivali)
led to the discovery that these rodents could regenerate 4 mm holes in
the ear pinna (Seifert et al., 2012) and subsequent research extended
these findings to include another species, A. cahirinus (Gawriluk et al.,
2016; Matias et al., 2015). Comprehensive studies in spiny mice and
rabbits have detailed the regeneration of full-thickness skin, hair
follicles, glands, adipose tissue, elastic cartilage, and to a limited
degree, muscle fibers (Fig. 3E) (Gawriluk et al., 2016; Joseph and
Dyson, 1966; Matias et al., 2015). That some species can regenerate ear
holes while others cannot make this model of epimorphic regeneration
attractive to uncover mechanisms that stimulate blastema formation
and differentiate a regenerative or fibrotic response to injury.
Superficially, the regenerative response follows the stereotypical
pattern observed during other examples of vertebrate appendage
regeneration. The initial trauma stimulates an injury response that
recruits neutrophils and monocytes to the injury site, while the
hemostatic response contributes to scab formation (Simkin et al.,
2017). Shortly thereafter, wound edge keratinocytes are stimulated to
migrate beneath the scab and re-epithelialize the injury (Gawriluk
et al., 2016; Joseph and Dyson, 1966; Seifert et al., 2012). Once the
epidermal barrier is restored, cells begin to accumulate centripetally
beneath the neoepidermis and as this accumulation continues a
regeneration blastema forms (Gawriluk et al., 2016; Joseph and
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Developmental Biology 433 (2018) 190–199
Dyson, 1966). Cell cycle re-entry, progression and proliferation are
observed in blastemal cells and a small group of epidermal cells at the
proximal wound margins (Gawriluk et al., 2016; Seifert et al., 2012). As
the ear hole is progressively filled, new tissue growth preferentially
occurs in the proximal portion of the hole (Fig. 3C-C′) and new tissue
growth is coincident with axon regeneration (Gawriluk et al., 2016) and
angiogenesis (Joseph and Dyson, 1966; Matias et al., 2015). Re-
differentiation of new tissues occurs outside to inside such that new
hair follicles and cartilage form nearer to the wound margin before they
appear in the center (Fig. 3C-D′) (Joseph and Dyson, 1966; Seifert
et al., 2012). Hair follicle regeneration in spiny mice is visible twenty
days post injury. As new tissue forms to close spiny mouse ear holes,
elastic cartilage regeneration occurs at two positions. Similar to
fracture healing, a callous forms at the cut end of the cartilage sheet
and this appears to contribute some portion of the new cartilage.
However, the majority of the new cartilage appears to emerge de novo
from mesenchymal condensations within the new tissue (Seifert et al.,
2012). Eventually, new tissue completely fills the hole and cellular
differentiation restores the excised tissue. These interconnected and
overlapping processes together constitute regeneration of a complex
musculoskeletal structure. Importantly, provided holes larger than
2 mm are made through the pinna, epimorphic regeneration of ear
holes can be differentiated from fibrosis-driven partial ear hole closure
observed in other rodents and laboratory mouse strains (Gawriluk
et al., 2016).
Whereas the digit tip provides a model to study regenerative
success and failure in the same organ (e.g., successful regeneration in
P3 versus regenerative failure in P2), the ear hole model of epimorphic
regeneration exploits interspecies variation in regenerative ability. In
contrast to regeneration observed in rabbits and spiny mice, broad
phylogenetic sampling shows that the ability to regenerate ear holes is
restricted among mammals (Gawriluk et al., 2016; Goss, 1980;
Williams-Boyce and Daniel, 1986). Ear hole regeneration may extend
to chinchillas, pikas and cats, but other rodents so far studied are
unable to regenerate ear holes. This raises the question as to how
regeneration occurs in some mammalian species? With the discovery of
regeneration in spiny mice, one approach has been to compare the
regenerative response of the ear pinna to fibrotic repair of the same
injury in other rodents. Comparing spiny mice to outbred Mus
musculus showed that formation and persistence of a blastema during
healing distinguishes the regenerative response from a scarring re-
sponse (Gawriluk et al., 2016; Seifert et al., 2012). This distinction
provides an opportunity to characterize a mammalian blastema and
tease apart cellular and molecular processes that operate during these
differential responses to injury.
Careful analysis of tissue regeneration in spiny mice has begun to
help define cellular and molecular features of the ear blastema
(Gawriluk et al., 2016; Matias et al., 2015; Seifert et al., 2012;
Simkin et al., 2017). As indicated above, a key characteristic of the
blastema is its epidermal compartment, the wound epidermis, which
forms following re-epithelialization. As mesenchymal cells accumulate
beneath the epidermis distal to the cut cartilage, epidermal cell
proliferation is excluded from the wound epidermis and maintained
in a small group of wound edge keratinocytes (Gawriluk et al., 2016).
Basal keratinocytes in this distal epidermis maintain a circular
morphology rather than assume the columnar morphology typical of
normal stratified epidermis and the distal epidermis observed in mice
during scarring. Importantly, keratin 17 is upregulated in the wound
epidermis and expressed until the ear hole is filled with new tissue, a
situation that does not occur during scarring where keratin 17
expression disappears by D15 post injury (Gawriluk et al., 2016).
These features are coincident with a failure to reconstitute the lamina
densa beneath the distal epidermis until hole closure is complete
(Gawriluk et al., 2016; Seifert et al., 2012). Together, these features
characterize the unique epidermal compartment of the blastema which
persists until hole closure is completed and the circular blastema
A.W. Seifert, K. Muneoka Developmental Biology 433 (2018) 190–199

Fig. 3. 4 mm ear punch assay and epimorphic regeneration in the spiny mouse Acomys cahirinus. (A) Position of a normal ear punch in an adult spiny mouse relative to the ear pinna
along the proximodistal (p/d) axis. (B) Masson's Trichrome stained tissue section of an uninjured spiny mouse ear pinna indicating: epidermis (e), dermis (d), adipose tissue (ad),
skeletal muscle (m), and elastic cartilage (c). Hair follicles and sebaceous glands are visible in the dorsal and ventral skin. Scale bar = 50 µm. (C-D) Whole mount images of regenerating
ear holes at D30 (C) and D40 (D) post injury along the proximodistal (p/d) axis. In regenerating ears growth is biased to the proximal part of the hole. White circles indicate original
4 mm punch. (C′-D′) Sections through C and D showing the regeneration blastema (C′) and ear hole closure (D′). New hair follicles, dermis, adipose tissue and cartilage are visible at
D40. Cartoon at right in C′ shows how tissue sections are collected. (E) A regenerated ear hole in Acomys cahirinus at D85 showing complete regeneration along the proximodistal and
dorsoventral axes. New muscle fibers are visible past the proximal amputation plane. Dotted black lines indicate the injury place (C′-E). Image in E modified slightly from Gawriluk et al.
(2016).

disappears. Further molecular characterization of the wound epidermis
in spiny mice and other vertebrates will help refine our understanding
of how this compartment regulates blastema formation and regenera-
tion.
Cells accumulating beneath the wound epidermis constitute the
mesenchymal compartment of the blastema, and another salient
feature of the blastema is the unique composition of the extracellular
matrix (ECM) that supports specific activities of blastemal cells during
morphogenesis. Fibrotic ECM is dominated by collagen with a ratio
favoring collagen 1 > collagen 3 (Gawriluk et al., 2016; Lo et al., 2012;
Marrero et al., 2017; Seifert and Maden, 2014; Simkin et al., 2015b).
Analysis of blastema ECM in comparison to matrix composition during
fibrotic healing revealed a profound difference in the extracellular
environment (Gawriluk et al., 2016; Seifert et al., 2012). While collagen
1 was produced during mammalian regeneration, it comprised only a
small fraction of the blastemal ECM, which was instead dominated by
fibronectin and tenascin-C among other matrix proteins (Gawriluk
et al., 2016). Fibronectin and tenascin-C promote cell migration and
proliferation, two cellular attributes required for morphogenesis during
regeneration. Beyond the capacity of ECM proteins to physically
interact with cells and stimulate behavior, the ECM also acts as a
reservoir for growth factors, cytokines and signaling molecules.
Although more work is required to fully characterize the blastemal
ECM in spiny mice, work across regenerating vertebrates strongly
supports ECM composition as a key marker of a regeneration blastema
(Calve et al., 2010; Gawriluk et al., 2016; Marrero et al., 2017; Onda
et al., 1991; Satoh et al., 2012; Seifert et al., 2012; Tassava et al., 1996).
While comparative transcriptomics may suggest that the blastema
extracellular environment has a unique molecular signature
(Gawriluk et al., 2016), the extent to which known and novel matrix
molecules are functionally required for blastema formation and
morphogenesis awaits testing.
Epimorphic regeneration in an adult occurs in the context of a fully
differentiated and operational immune system. Prior to blastema
formation, local injury elicits an immune response. Although innate
and adaptive immune defenses occur in both scarring and regenerating
systems, recent work from mammals and other vertebrates suggests
that aspects of these responses may positively regulate a regenerative
response (Gauron et al., 2013; Godwin et al., 2013; Lai et al., 2017;
Love et al., 2013; Petrie et al., 2014; Simkin et al., 2017). Comparing
the inflammatory response during regeneration and scarring in spiny
mice revealed that reactive oxygen species produced by NAPDH-
oxidase at the injury site were significantly elevated during regenera-
tion and these molecules persisted in elevated amounts during
blastema formation (Simkin et al., 2017). This mirrors a proposed role
for reactive oxygen species during tail regeneration in Xenopus and

196
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zebrafish (Gauron et al., 2013; Love et al., 2013) and brain regenera-
tion in newts (Hameed et al., 2015). Macrophages are a key regulator of
the local immune response and vertebrate studies suggests that they
control inflammation, fibrosis and blastema formation during regen-
eration (Godwin et al., 2013; Petrie et al., 2014; Simkin et al., 2017).
Depleting macrophages during normal fibrotic wound healing demon-
strated they control the rate of re-epithelialization, fibrosis and cell
proliferation (Leibovich and Ross, 1975; Mirza et al., 2009). Similarly,
macrophages infiltrate the ear pinna and are present during regenera-
tion and scarring. Depleting macrophages in the ear pinna prior to and
during injury demonstrated a functional requirement for these cells
during ear hole regeneration (Simkin et al., 2017). Interestingly,
depleting macrophages delayed re-epithelialization and inhibited blas-
tema formation, and when macrophages were allowed to infiltrate the
ear pinna a blastema formed and regeneration ensued. Beyond the
assessment that macrophages are generally required for epimorphic
regeneration, some evidence suggests specific macrophage subtypes
may differentially regulate this response to injury (Mescher, 2017;
Simkin et al., 2017). Interestingly, when macrophage subtypes were
analyzed during spiny mouse ear hole regeneration, classically acti-
vated (M1) macrophages were largely absent from the blastema
(Simkin et al., 2017). This study left open whether the blastema
triggered macrophage phenotype switching or instead certain subtypes
were restricted from infiltrating the blastema. Further studies will need
to determine the importance of particular macrophage subtypes and
the extent to which these subtypes may regulate blastema formation
either by directly stimulating local fibroblasts or indirectly by polariz-
ing the immune response.
The identity and plasticity of progenitor cells that form the ear
blastema is currently unknown. The mesenchymal compartment of the
ear pinna develops entirely from Hoxa2-expressing cranial neural crest of
the second pharyngeal arch, whereas the epidermal compartment is
derived from ectoderm (Minoux et al., 2013). Whereas subsequent
differentiation of these neural crest progenitors during external ear
development is less well understood, Hoxa2 regulates BMP signaling,
specifically Bmp5 and Bmp4, during cartilage development in the pinna
(Minoux et al., 2013). While the prevailing view of the cellular contribu-
tion to the blastema is that lineage restricted cells will supply progenitors
to a heterogeneous pool that will expand and differentiate into specific
tissue subsets, what remains unknown is the degree to which lineage
restriction applies to local cells that contribute to the ear blastema. For
instance, if the shared cranial crest cell origin of all non-epidermal tissue
structures in the ear pinna sets the plasticity limit during regeneration,
then in theory, the pool of available cells for regeneration would extend to
all mesenchymal cells of the ear. Similarly, if regeneration in the ear is
dependent on reserves of adult stem cells rather than some form of de-
differentiation then fewer populations would be necessary to replace all
the missing tissues in the ear. A different question is whether the
appropriate cells are present for regeneration, but they are simply unable
to form a blastema. In this scenario, heterogeneous blastemal cells that
normally contribute to regeneration in spiny mice are resistant to cell
cycle re-entry, progression and division in non-regenerating mammals.
Evidence to support the latter comes from research showing that while the
number of Ki67+ cells in the ear during regeneration and scarring is
similar, cell cycle progression and division is rarely observed during
scarring (Gawriluk et al., 2016). This result was partly attributed to the
observation that p21 and p27 were found throughout cell nuclei during
scarring, but were absent from the blastema during regeneration.
Importantly, the blastema in spiny mice was significantly enriched with
markers for the G1/S transition (pRb), DNA replication (EdU) and
mitosis (PHH3) which differentiated blastema cells from cells present
during fibrotic healing in lab mice (Gawriluk et al., 2016). Understanding
whether or not cell proliferation occurs in response to specific paracrine
signals or results from an intrinsic ability to undergo cell cycle progression
will be vital in discovering how spiny mice form an ear blastema and
undergo regeneration.
197
Developmental Biology 433 (2018) 190–199
4. Conclusions: expanding the limits of regeneration
The continued study of epimorphic regeneration in mammals has
much to add to our understanding of vertebrate regeneration
biology. The ear hole model discussed above provides an experi-
mental system to uncover key mechanisms that explain differences
in regenerative ability across mammalian species. When applied
more broadly, it may also help identify additional mammalian
species that possess enhanced regenerative ability. As a comparative
system between regenerating and non-regenerating species, the ear
punch assay will reveal whether specific molecules or pathways can
serve as early indicators for blastema formation and which factors
alone or in combination are required to maintain blastema morpho-
genesis. This will be necessary if we hope to uncover potential
targets that might be used to stimulate regeneration. In this light,
both the ear hole and digit tip models offer an added translational
perspective to the biology of regeneration. In digit studies, amputa-
tion of the mouse P2 digit has become a useful regeneration
incompetent model to test strategies for inducing a regeneration
response (Dawson et al., 2016). BMP signaling has been shown to be
important for digit tip regeneration (Yu et al., 2010) and digit
amputation at a P2 level is stimulated to regenerate a patterned
response by targeted BMP2 treatment in both neonates and adult
mice (Dawson et al., 2017; Yu et al., 2012). The BMP2 response
indirectly induces the recruitment of cells to the amputation wound
by activating CXCL12 production (Lee et al., 2013) and BMP2
functions as a mitogen for chondrocytes that establish an endochon-
dral ossification center at the amputation wound (Dawson et al.,
2017; Yu et al., 2012). Once formed, the induced endochondral
ossification center organizes the morphogenesis of new distal
skeletal tissue that replaces the amputated P2 bone. These studies
provide proof of concept that a regenerative response can be
stimulated at a regeneration incompetent amputation wound, and
when combined with results from the ear leads to two important
conclusions. First, progenitor cell availability is not a limiting factor
at a mammalian amputation injury. Indeed, it seems unlikely that
different progenitor cell populations would be present in the second
and third phalanx of the digit or in the ear pinna from different
rodent species. Second, limitation of the wound microenvironment
to activate progenitor cells is in part responsible for regenerative
failure. How exactly this activation is controlled remains the focus of
current and future investigations. As studies using mammalian
regeneration models progress they will continue providing insight
into how injured tissue can be transformed into a regeneration
blastema and how blastemal morphogenesis is controlled to appro-
priately replace injured tissue in situ. This will require dissecting
components of the blastema to uncover new characteristics or
markers that can indicate when these two transformations take
place and using these markers to test approaches at successfully
stimulating regeneration where it does not normally occur.
Acknowledgements
We would like to thank Jennifer Simkin and two anonymous
reviewers for comments and critical reading of the manuscript and
Ricardo Zayas and Karen Echeverri for soliciting our review. A.W.
Seifert is supported by the National Science Foundation (NSF) and the
Office for International Science and Engineering (OISE) (IOS
-1353713) and the National Institute of Arthritis and
Musculoskeletal and Skin Diseases (NIAMS – NIH) (R01AR070313).
K. Muneoka is supported by Texas A & M University.
Competing interests
The authors declare no competing interests.
A.W. Seifert, K. Muneoka
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